The invention relates generally to the field of nucleic amplification reactions and more particularly to a device that rapidly and economically amplifies, detects and measures polynucleotide products from nucleic acid amplification processes, such as polymerase chain reaction.
Nucleic acid sequence analysis using polymerase chain reaction (PCR) and other nucleic acid amplification techniques has been in the forefront of the rapid expansion of molecular testing and research worldwide. The development of several nucleic acid amplification technologies has played a major role in this rapid expansion of nucleic acid analysis. A variety of instrumentation has been developed to perform nucleic acid analysis, the most widespread being PCR technology.
The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al., describe the basics of what has become PCR technology for increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification.
PCR technology or methodology has become the de facto standard for the amplification of nucleic acid. The most ubiquitous nucleic acid amplification systems that have been developed to perform PCR and other nucleic amplification techniques are comprised of thermal cycler instruments that have as their major component a thermal conductive block of material that alternately heats and cools a thermal conductive container, usually made of glass or plastic, placed on it. This container holds a fluid sample mixture containing the targeted genetic specimen material and reagents (in the case of a PCR amplification the fluid sample contains nucleic acid material, thermostable DNA polymerase and primers designed with sequences complementary to the targeted nucleic acid). The primers may contain at the 5′ end may contain a convenient reporter molecule such as radioisotope, biotin, floriscein, etc. During a PCR reaction, the thermal cycler instrument alternately heats and cools the thermal conductive block on which the sample material is in contact; cycling the sample mixtures first to a temperature of approximately 95° C., causing the denaturation of the double-stranded then cooling it to a temperature approximately 55° C. allowing the primers in the sample mixture to anneal to the resulting single-stranded templates from the specimen and then heating the sample mixture to approximately 72° C. where the thermostable DNA polymerase synthesizes a new strand of DNA from the extension of the primer annealed to the template complementary to that of the single-stranded DNA template creating 2 new double-stranded DNA pairs. The thermal cycler then continues to cycle the sample mixture through the 95° C. to 55° C. to 72° temperatures replicating the denaturation, annealing and synthesis processes and doubling at each cycle the targeted DNA before it is fully amplified resulting in the amplification of the original genetic material or DNA fragment to over 109 its original number. The amplified material is then removed from the thermal cycler placed in an instrument or on a device containing a probe complementary to the targeted material that will fluoresce in proportion to the amount of targeted material in the amplified sample.
Newer models of thermal cycler instruments have built-in detection apparatus that automatically detect the presence of the targeted genetic material once it is amplified.
Critical to the successful PCR amplification of sample material is the heating and cooling of the sample to the required temperatures, the presence of a sufficient amount of thermostable DNA polymerase needed to synthesize new DNA strands plus a large excess of primers, so that the two strands will always bind to the primers, instead of with each other, and a reaction that is carried out in a closed or sealed reaction environment that prevents cross contamination or sample carryover. Without these conditions being met it is highly unlikely that the thermal cycle amplification process will be successful.
Recent advances in thermal cycler technology have resulted in air-based temperature control using hot air jets to rapidly heat and cool the sample material or heating and cooling the sample materials in microfluidic chambers, thereby replacing the thermal conductive block system. Some of these newer technologies continuously or at various times during the amplification detect or identify the targeted amplified genetic material.
These advances in prior art thermal cycler technology have not alleviated all of the problems with the prior art. There still exists a need to improve prior art that while significantly reducing the time required to amplify nucleic acid material down to thirty (30) minutes still require highly trained technicians to operate, are largely fixed immobile instruments that weigh from 12 to 25 kilograms, remain subject to cross contamination and sample carryover and result in a significant increase in the thermal cycler system cost of ownership and an increase in the reagent cost per analysis.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned as well as those inherent therein and represents a significant advancement over prior art.